Targeted chromosomal mutagenasis using zinc finger nucleases

ABSTRACT

The present invention provides for a method or methods of targeted genetic recombination or mutagenesis in a host cell or organism, and compositions useful for carrying out the method. The targeting method of the present invention exploits endogenous cellular mechanisms for homologous recombination and repair of double stranded breaks in genetic material. The present invention provides numerous improvements over previous mutagenesis methods, such advantages include that the method is generally applicable to a wide variety of organisms, the method is targeted so that the disadvantages associated with random insertion of DNA into host genetic material are eliminated, and certain embodiments require relatively little manipulation of the host genetic material for success. Additionally, it provides a method that produces organisms with specific gene modifications in a short period of time.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. Provisional PatentApplication No. 60/351,035 filed Jan. 23, 2002 and from PCT ApplicationNo. PCT/US03/02012 filed Jan. 22, 2003.

ACKNOWLEDGMENT OF FEDERAL RESEARCH SUPPORT

The U.S. Government has certain rights in the invention based uponpartial support by Grant RO1 GM 58504.

BACKGROUND OF THE INVENTION

Gene targeting—the process of gene replacement or mutation by homologousrecombination—is a very useful, but typically inefficient technique forintroducing desired changes in the genetic material of a host cell. Onlywhen powerful selection for the targeted product can be applied isrecovery of the desired alteration possible. A general method forimproving the efficiency of gene targeting would be valuable in manycircumstances, as would extension of this tool to a broader range oforganisms.

It has been demonstrated in model experiments that introduction of adouble-strand break (DSB) in host DNA greatly enhances the frequency oflocalized recombination. However, those tests required insertion of arecognition site for a specific endonuclease before cleavage could beinduced. Similarly, in Drosophila the DSBs produced by P-elementexcision are recombinagenic, but require the P-element to preexist atthe target site.

Previously demonstrated methods of genetic transformation have beenhighly successful; however transformation without targeted recombinationhas been accompanied by problems associated with random insertion of theintroduced DNA. Random integration can lead to the inactivation ofessential genes, or to the aberrant expression of the introduced gene.Additional problems associated with genetic transformation includemosaicism due to multiple integrations.

Targeted genetic recombination or mutation of a cell or organism is nowdesirable because complete genomic sequences have been determined for anumber of organisms, and more sequences are being obtained each day. Notonly would the ability to direct a mutation to a specific genetic locusgreatly aid those studying the function of particular genes, buttargeted genetic recombination would also have therapeutic andagricultural applications. Methods of targeted genetic recombination areneeded that are more general, efficient, and/or reproducible thancurrently available techniques.

SUMMARY OF THE INVENTION

The present invention provides compositions and methods for carrying outtargeted genetic recombination or mutation. Any segment of endogenousnucleic acid in a cell or organism can be modified by the method of theinvention as long as the sequence of the target region, or portion ofthe target region, is known, or if isolated DNA homologous to the targetregion is available.

In certain embodiments, the compositions and methods comprise thetransformation of a host organism by introducing a nucleic acid moleculeencoding a chimeric zinc finger nuclease (ZFN) into a cell or organismand identifying a resulting cell or organism in which a selectedendogenous DNA sequence is cleaved and exhibits a mutation.

In a preferred embodiment, such methods comprise selecting a zinc fingerDNA binding domain capable of preferentially binding to a specific hostDNA locus to be mutated; further selecting a non-specific DNA cleavagedomain capable of cleaving double-stranded DNA when operatively linkedto said binding domain and introduced into the host cell; furtherselecting a promoter region capable of inducing expression in the hostcell; and further operatively linking DNA encoding the binding domainand the cleavage domain and the promoter region to produce a DNAconstruct. The DNA construct is then introduced into a target host celland at least one host cell exhibiting recombination at the target locusin the host DNA is identified. In a particular embodiment, the DNAbinding domain comprises the binding domains of three Cis₂His₂ zincfingers. In another embodiment, the cleavage domain comprises a cleavagedomain derived from the Type II restriction endonuclease FokI. In oneembodiment, an inducible heat shock promoter is operatively linked toDNA encoding the chimeric zinc finger nuclease.

Additional embodiments involve methods for targeted insertion byhomologous recombination of selected DNA sequences (donor DNA). DonorDNA can comprise a sequence that encodes a product to be produced in thehost cell. Said product can be a product produced for the benefit of thehost cell or organism (for example, gene therapy), or the product can beone that is produced for use outside the host cell or organism (forexample, the product may be selected from, but not limited to,pharmaceuticals, hormones, protein products used in the manufacture ofuseful objects or devices, nutriceuticals, products used in chemicalmanufacture or synthesis, etc.).

In a certain embodiment, the present invention is utilized to disrupt atargeted gene in a somatic cell. Such gene may be over-expressed in oneor more cell types resulting in disease. Disruption of such gene mayonly be successful in a low percentage of somatic cells but suchdisruption may contribute to better health for an individual sufferingfrom disease due to over-expression of such gene.

In another embodiment, the present invention can be utilized to disrupta targeted gene in a germ cell. Cells with such disruption in thetargeted gene can be selected for in order to create an organism withoutfunction of the targeted gene. In such cell the targeted gene functioncan be completely knocked out.

In another embodiment, the present invention can be utilized to enhanceexpression of a particular gene by the insertion of a control elementinto a somatic cell. Such a control element may be selected from a groupconsisting of, but not limited to, a constitutively active, inducible,tissue-specific or development stage-specific promoters. Such controlelement may be targeted to a chromosomal locus where it will effectexpression of a particular gene that is responsible for a product with atherapeutic effect in such a cell or the host organism. The presentinvention may further provide for the insertion of donor DNA containinga gene encoding a product that, when expressed, has a therapeutic effecton the host cell or organism. An example of such a therapeutic methodwould be to use the targeted genetic recombination of the presentinvention to effect insertion into a pancreatic cell of an activepromoter operatively linked to donor DNA containing an insulin gene. Thepancreatic cell containing the donor DNA would then produce insulin,thereby aiding a diabetic host. Additionally, donor DNA constructs couldbe inserted into a crop genome in order to effect the production of apharmaceutically relevant gene product. A gene encoding apharmaceutically useful protein product, such as insulin or hemoglobin,functionally linked to a control element, such as a constitutivelyactive, inducible, tissue-specific or development stage-specificpromoter, could be inserted into a host plant in order to produce alarge amount of the pharmaceutically useful protein product in the hostplant. Such protein products could then be isolated from the plant.Alternatively, the above-mentioned methods can be utilized in a germcell.

The present invention can be utilized in both somatic and germ linecells to effect alteration at any chromosomal target locus.

Methods of the present invention are applicable to a wide range of celltypes and organisms. The present invention can apply to any of thefollowing cells, although the methods of the invention are not limitedto the cells or organisms herein listed: A single celled ormulticellular organism; an oocyte; a gamete; a germline cell in cultureor in the host organism; a somatic cell in culture or in the hostorganism; an insect cell, including an insect selected from the groupconsisting of Coleoptera, Diptera, Hemiptera, Homoptera, Hymenoptera,Lepidoptera, or Orthoptera, including a fruit fly, a mosquito and amedfly; a plant cell, including a monocotyledon cell and a dicotyledoncell; a mammalian cell, including but not limited to a cell selectedfrom the group consisting of mouse, rat, pig, sheep, cow, dog or catcells; an avian cell, including, but not limited to a cell selected fromthe group consisting of chicken, turkey, duck or goose cells; or a fishcell, including, but not limited to zebrafish, trout or salmon cells.

Many alterations and variations of the invention exist as describedherein. The invention is exemplified for targeted genetic recombinationin the insect, Drosophila and the plants, Arabidopsis and tobacco. InDrosophila and Arabidopsis, the nucleotide sequence is known for most ofthe genome. Large segments of genomic sequences from other organisms arebecoming known at a fast pace. The elements necessary to carry out themethods of the present invention as herein disclosed can be adapted forapplication in any cell or organism. The invention therefore provides ageneral method for targeted genetic recombination in any cell ororganism.

Table 1: Illustrates the number of germline mutants recovered bycrossing males exposed to a heat shock with attached-X [C(1)DX] femalesand females from the heat shock to FM6 (y) males in accordance with anembodiment of the present invention. The percent of all the heat-shockedparents screened that gave at least one germline mutant is shown inparentheses in the # Giving y column. The total number of mutant fliesrecovered is given in the Total y column and also expressed as a percentof all candidate offspring (in parentheses). The number of mutantoffspring per fly varied from 1 to 15.

Table 2: Illustrates the efficiency of ZFN-mediated recombination intobacco protoplasts relative to controls. Protoplasts were tested forkanamycin resistance (Kan^(r)) and β-glucuronidase activity (GUS⁺).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods and compositions for carryingout targeted genetic recombination or mutation. In contrast topreviously known methods for targeted genetic recombination, the presentinvention is efficient and inexpensive to perform and is adaptable toany cell or organism. Any segment of double-stranded nucleic acid of acell or organism can be modified by the method of the present invention.The method exploits both homologous and non-homologous recombinationprocesses that are endogenous in all cells.

The method of the present invention provides for both targeted DNAinsertions and targeted DNA deletions. The method involvestransformation of a cell with a nucleic acid construct minimallycomprising DNA encoding a chimeric zinc finger nuclease (ZFN). In aparticular embodiment, the method further involves transforming a cellwith a nucleic acid construct comprising donor DNA. Other schemes basedon these general concepts are within the scope and spirit of theinvention, and are readily apparent to those skilled in the art.

The present invention can be utilized in both somatic and germ cells toconduct genetic manipulation at a particular genetic locus.

In a particular embodiment, the present invention is utilized to disrupta gene in a somatic cell wherein that gene is over-expressing a productand/or expressing a product that is deleterious to the cell or organism.Such gene may be over-expressed in one or more cell types resulting indisease. Disruption of such gene by the methods of the present inventionmay contribute to better health for an individual suffering from diseasedue to expression of such gene. In other words, disruption of genes ineven a small percentage of cells can work to decrease expression levelsin order to produce a therapeutic effect.

In another embodiment, the present invention can be utilized to disrupta gene in a germ cell. Cells with such disruption in a particular genecan be selected for in order to create an organism without function ofsuch gene. In such a cell the gene can be completely knocked-out. Theabsence of function in this particular cell can have a therapeuticeffect, or in the case of plants, can create a new phenotype.

In another embodiment, the present invention can be utilized to enhanceexpression of a particular gene by the insertion of a control elementinto a somatic cell. Such control element may be a constitutivelyactive, inducible or developmental stage-specific promoter. It may alsobe a tissue-specific promoter capable of effecting expression only inparticular cell types. Such control element may be placed in such amanner to effect expression of a particular gene that is responsible fora product with a therapeutic effect in such a cell.

The present invention may further provide for the insertion of donor DNAencoding a gene product that, when constitutively expressed, has atherapeutic effect. An example of this embodiment would be to insertsuch DNA constructs into an individual suffering from diabetes in orderto effect insertion of an active promoter and donor DNA encoding theinsulin gene in a population of pancreatic cells. This population ofpancreatic cells containing the exogenous DNA would then produceinsulin, thereby aiding the diabetic patient. Additionally, such DNAconstructs could be inserted into crops in order to effect theproduction of pharmaceutically-relevant gene products. Genes for proteinproducts, such as insulin, lipase or hemoglobin, could be inserted intoplants along with control elements, such as constitutively active orinducible promoters, in order to produce large amounts of thesepharmaceuticals in a plant. Such protein products could then be isolatedfrom the plant. Transgenic plants or animals may be produced with thesegenetic alterations. Tissue or cell-type specific vectors may also beemployed for providing gene expression only in the cells of choice.

Alternatively, the above-mentioned methods can be utilized in a germcell in order to select cells where insertion has occurred in theplanned manner in order for all subsequent cell divisions to producecells with the desired genetic change. For example, site-specific DNAinsertion, deletion, or modification of plant coding sequences mayconfer new traits to plants, such as resistance to disease or pests.

As used herein, the cells in which genetic manipulation occurs and anexogenous DNA segment or gene has been introduced through the hand ofman are called recombinant cells. Therefore, recombinant cells aredistinguishable from naturally occurring cells which do not contain arecombinantly introduced exogenous DNA segment or gene. Recombinantcells include those having an introduced cDNA or genomic gene, and alsoinclude genes positioned adjacent to a heterologous promoter notnaturally associated with the particular introduced gene.

To express a recombinant encoded protein or peptide, whether mutant orwild-type, in accordance with the present invention one would prepare anexpression vector that comprises isolated nucleic acids under thecontrol of, or operatively linked to, one or more promoters, which maybe inducible, constitutively active or tissue specific, for example. Tobring a coding sequence “under the control of” a promoter, one positionsthe 5′ end of the transcription initiation site of the transcriptionalreading frame generally between about 1 and about 50 nucleotides“downstream” (i.e., 3′) of the chosen promoter. The “upstream” promoterstimulates transcription of the DNA and promotes expression of theencoded recombinant protein. This is the meaning of “recombinantexpression” in this context.

Ways of effecting protein expression are well known in the art. Oneskilled in the art is capable of expressing a protein of his or herchoice in accordance with the present invention.

The methods of the present invention can be applied to whole organismsor in cultured cells or tissues or nuclei, including those cells,tissues or nuclei that can be used to regenerate an intact organism, orin gametes such as eggs or sperm in varying stages of their development.Because DSBs stimulate mutagenic repair in essentially all cells ororganisms, cleavage by ZFNs may be used in any cells or organisms. Themethods of the present invention can be applied to cells derived fromany organism, including but not limited to insects, fungi, rodents,cows, sheep, goats, chickens, and other agriculturally importantanimals, as well as other mammals, including, but not limited to dogs,cats and humans.

Additionally, the compositions and methods of the present invention maybe used in plants. It is contemplated that the compositions and methodscan be used in any variety of plant species, including monocots ordicots. In certain embodiments, the invention can be used in plants suchas grasses, legumes, starchy staples, Brassica family members, herbs andspices, oil crops, ornamentals, woods and fibers, fruits, medicinalplants, poisonous plants, corn, cotton, castor bean and any other cropspecie. In alternative embodiments, the invention can be used in plantssuch as sugar cane, wheat, rice, maize, potato, sugar beet, cassava,barley, soybean, sweet potato, oil palm fruit, tomato, sorghum, orange,grape, banana, apple, cabbage, watermelon, coconut, onion, cottonseed,rapeseed and yam. In some embodiments, the invention can be used inmembers of the Solanaceae specie, such as tobacco, tomato, potato andpepper. In other embodiments, the invention can be used in poisonousornamentals, such as oleander, any yew specie and rhododendron. In aparticular embodiment, the Brassica specie is Arabidopsis. In anotherembodiment, the plant species is tobacco.

Grasses include, but are not limited to, wheat, maize, rice, rye,triticale, oats, barley, sorghum, millets, sugar cane, lawn grasses andforage grasses. Forage grasses include, but are not limited to, Kentuckybluegrass, timothy grass, fescues, big bluestem, little bluestem andblue gamma. Legumes include, but are not limited to, beans like soybean,broad or Windsor bean, kidney bean, lima bean, pinto bean, navy bean,wax bean, green bean, butter bean and mung bean; peas like green pea,split pea, black-eyed pea, chick-pea, lentils and snow pea; peanuts;other legumes like carob, fenugreek, kudzu, indigo, licorice, mesquite,copaifera, rosewood, rosary pea, senna pods, tamarind, and tuba-root;and forage crops like alfalfa. Starchy staples include, but are notlimited to, potatoes of any species including white potato, sweetpotato, cassava, and yams. Brassica, include, but are not limited to,cabbage, broccoli, cauliflower, brussel sprouts, turnips, collards, kaleand radishes. Oil crops include, but are not limited to, soybean, palm,rapeseed, sunflower, peanut, cottonseed, coconut, olive palm kernel.Woods and fibers include, but are not limited to, cotton, flax, andbamboo. Other crops include, but are not limited to, quinoa, amaranth,tarwi, tamarillo, oca, coffee, tea, and cacao.

Definitions:

For the purposes of the present invention, the following terms shallhave the following meanings:

As used herein, the term “targeted genetic recombination” refers to aprocess wherein recombination occurs within a DNA target locus presentin a host cell or host organism. Recombination can involve eitherhomologous or non-homologous DNA. One example of homologous targetedgenetic recombination would be cleavage of a selected locus of host DNAby a zinc finger nuclease (ZFN), followed by homologous recombination ofthe cleaved DNA with homologous DNA of either exogenous or endogenousorigin. One example of non-homologous targeted genetic recombinationwould be cleavage of a selected locus of host DNA by a ZFN, followed bynon-homologous end joining (NHEJ) of the cleaved DNA.

As used herein, the terms “host cell” or “host organism” or, simply,“target host”, refer to a cell or an organism that has been selected tobe genetically transformed to carry one or more genes for expression ofa function used in the methods of the present invention. A host canfurther be an organism or cell that has been transformed by the targetedgenetic recombination or mutation methods of the present invention.

The term “target” or “target locus” or “target region” refers herein tothe gene or DNA segment selected for modification by the targetedgenetic recombination method of the present invention. Ordinarily, thetarget is an endogenous gene, coding segment, control region, intron,exon or portion thereof, of the host organism. However, the target canbe any part or parts of the host DNA.

For the purposes of the present invention, the term “zinc fingernuclease” or “ZFN” refers to a chimeric protein molecule comprising atleast one zinc finger DNA binding domain effectively linked to at leastone nuclease capable of cleaving DNA. Ordinarily, cleavage by a ZFN at atarget locus results in a double stranded break (DSB) at that locus.

For the purposes of the present invention, the term “marker” refers to agene or sequence whose presence or absence conveys a detectablephenotype to the host cell or organism. Various types of markersinclude, but are not limited to, selection markers, screening markersand molecular markers. Selection markers are usually genes that can beexpressed to convey a phenotype that makes an organism resistant orsusceptible to a specific set of environmental conditions. Screeningmarkers can also convey a phenotype that is a readily observable anddistinguishable trait, such as Green Fluorescent Protein (GFP), GUS orbeta-galactosidase. Molecular markers are, for example, sequencefeatures that can be uniquely identified by oligonucleotide probing, forexample RFLP (restriction fragment length polymorphism), or SSR markers(simple sequence repeat).

As used herein, the term “donor” or “donor construct” refers to theentire set of DNA segments to be introduced into the host cell ororganism as a functional group. The term “donor DNA” as used hereinrefers to a DNA segment with sufficient homology to the region of thetarget locus to allow participation in homologous recombination at thesite of the targeted DSB.

For the purposes of the present invention, the term “gene” refers to anucleic acid sequence that includes the translated sequences that encodea protein (“exons”), the untranslated intervening sequences (“introns”),the 5′ and 3′ untranslated region and any associated regulatoryelements.

For the purposes of the present invention, the term “sequence” means anyseries of nucleic acid bases or amino acid residues, and may or may notrefer to a sequence that encodes or denotes a gene or a protein. Many ofthe genetic constructs used herein are described in terms of therelative positions of the various genetic elements to each other. Forthe purposes of the present invention, the term “adjacent” is used toindicate two elements that are next to one another without implyingactual fusion of the two elements. Additionally, for the purposes of thepresent invention, “flanking” is used to indicate that the same,similar, or related sequences exist on either side of a given sequence.Segments described as “flanking” are not necessarily directly fused tothe segment they flank, as there can be intervening, non-specified DNAbetween a given sequence and its flanking sequences. These and otherterms used to describe relative position are used according to normalaccepted usage in the field of genetics.

For the purposes of the present invention, the term “recombination” isused to indicate the process by which genetic material at a given locusis modified as a consequence of an interaction with other geneticmaterial. For the purposes of the present invention, the term“homologous recombination” is used to indicate recombination occurringas a consequence of interaction between segments of genetic materialthat are homologous, or identical. In contrast, for purposes of thepresent invention, the term “non-homologous recombination” is used toindicate a recombination occurring as a consequence of interactionbetween segments of genetic material that are not homologous, oridentical. Non-homologous end joining (NHEJ) is an example ofnon-homologous recombination.

For the purposes of the present invention, the term “nutriceutical”shall refer to any substance that is a food or part of a food andprovides medical or health benefits, including the prevention andtreatment of disease. Exemplary “nutraceuticals” include isolatednutrients, dietary supplements, herbal products and the like.

Moreover, for the purposes of the present invention, the term “a” or“an” entity refers to one or more than one of that entity; for example,“a protein” or “an nucleic acid molecule” refers to one or more of thosecompounds, or at least one compound. As such, the terms “a” or “an”,“one or more” and “at least one” can be used interchangeably herein. Itis also to be noted that the terms “comprising,” “including,” and“having” can be used interchangeably. Furthermore, a compound “selectedfrom the group consisting of” refers to one or more of the compounds inthe list that follows, including mixtures (i.e. combinations) of two ormore of the compounds. According to the present invention, an isolatedor biologically pure compound is a compound that has been removed fromits natural milieu. As such, “isolated” and “biologically pure” do notnecessarily reflect the extent to which the compound has been purified.An isolated compound of the present invention can be obtained from itsnatural source, can be produced using molecular biology techniques orcan be produced by chemical synthesis.

Zinc Finger Nucleases

A zinc finger nuclease (ZFN) of the present invention is a chimericprotein molecule capable of directing targeted genetic recombination ortargeted mutation in a host cell by causing a double stranded break(DSB) at the target locus. A ZFN of the present invention includes aDNA-binding domain and a DNA-cleavage domain, wherein the DNA bindingdomain includes at least one zinc finger and is operatively linked to aDNA-cleavage domain. The zinc finger DNA-binding domain is at theN-terminus of the chimeric protein molecule and the DNA-cleavage domainis located at the C-terminus of said molecule.

A ZFN as herein described must have at least one zinc finger. In apreferred embodiment a ZFN of the present invention would have at leastthree zinc fingers in order to have sufficient specificity to be usefulfor targeted genetic recombination in a host cell or organism. A ZFNcomprising more than three zinc fingers is within the scope of theinvention. A ZFN having more than three zinc fingers, although moretime-consuming to construct, would have progressively greaterspecificity with each additional zinc finger. In a particularembodiment, the DNA-binding domain is comprised of three zinc fingerpeptides operatively linked to a DNA cleavage domain.

The zinc finger domain of the present invention can be derived from anyclass or type of zinc finger. In a particular embodiment, the zincfinger domain comprises the Cys₂His₂ type of zinc finger that is verygenerally represented, for example, by the zinc finger transcriptionfactors TFIIIA or Sp1. In a preferred embodiment, the zinc finger domaincomprises three Cys₂His₂ type zinc fingers.

The DNA recognition and/or the binding specificity of a ZFN can bealtered in order to accomplish targeted genetic recombination at anychosen site in cellular DNA. Such modification can be accomplished usingknown molecular biology and/or chemical synthesis techniques. ZFNscomprising zinc fingers having a wide variety of DNA recognition and/orbinding specificities are within the scope of the present invention.

The ZFN DNA-cleavage domain is derived from a class of non-specific DNAcleavage domains, for example the DNA-cleavage domain of a Type IIrestriction enzyme. In a particular embodiment the DNA-cleavage domainis derived from the Type II restriction enzyme, FokI.

In a particular embodiment, a ZFN comprises three Cys₂His₂ type zincfingers, and a DNA-cleavage domain derived from the Type II restrictionenzyme, FokI. According to this embodiment, each zinc finger contacts 3consecutive base pairs of DNA creating a 9 bp recognition sequence forthe ZFN DNA binding domain. The DNA-cleavage domain of the embodimentrequires dimerization of two ZFN DNA-cleavage domains for effectivecleavage of double-stranded DNA. This imposes a requirement for twoinverted recognition (target DNA) sites within close proximity foreffective targeted genetic recombination. If all positions in the targetsites are contacted specifically, these requirements enforce recognitionof a total of 18 base pairs of DNA. There may be a space between the twosites. In a particular embodiment, the space between recognition sitesfor ZFNs of the present invention may be equivalent to 6 to 35 bp ofDNA. The region of DNA between the two recognitions sites is hereinreferred to as the “spacer”.

A linker, if present, between the cleavage and recognition domains ofthe ZFN comprises a sequence of amino acid residues selected so that theresulting linker is flexible. Or, for maximum target site specificity,linkerless constructs are made. A linkerless construct has a strongpreference for binding to and then cleaving between recognition sitesthat are 6 bp apart. However, with linker lengths of between 0 and 18amino acids in length, ZFN-mediated cleavage occurs between recognitionsites that are between 5 and 35 bp apart. For a given linker length,there will be a limit to the distance between recognition sites that isconsistent with both binding and dimerization. In a particularembodiment, there is no linker between the cleavage and recognitiondomains, and the target locus comprises two nine nucleotide recognitionsites in inverted orientation with respect to one another, separated bya six nucleotide spacer.

In order to target genetic recombination or mutation according to aparticular embodiment of the present invention, two 9 bp zinc finger DNArecognition sequences must be identified in the host DNA. Theserecognition sites will be in an inverted orientation with respect to oneanother and separated by about 6 bp of DNA. ZFNs are then generated bydesigning and producing zinc finger combinations that bind DNAspecifically at the target locus, and then linking the zinc fingers to acleavage domain of a Type II restriction enzyme.

Targeted Genetic Recombination or Mutation

The method of the present invention can be used for targeted geneticrecombination or mutation of any cell or organism. Minimum requirementsinclude a method to introduce genetic material into a cell or organism(either stable or transient transformation), sequence informationregarding the endogenous target region, and a ZFN construct orconstructs that recognizes and cleaves the target locus. According tosome embodiments of the present invention, for example homologousrecombination, donor DNA may also be required.

According to another embodiment of the present invention, DNA encodingan identifiable marker will also be included with the DNA construct.Such markers may include a gene or sequence whose presence or absenceconveys a detectable phenotype to the host cell or organism. Varioustypes of markers include, but are not limited to, selection markers,screening markers and molecular markers. Selection markers are usuallygenes that can be expressed to convey a phenotype that makes an organismresistant or susceptible to a specific set of environmental conditions.Screening markers can also convey a phenotype that is a readilyobservable and distinguishable trait, such as Green Fluorescent Protein(GFP), beta-glucuronidase (GUS) or beta-galactosidase. Markers may alsobe negative or positive selectable markers. In a particular embodiment,such negative selectable marker is codA. Molecular markers are, forexample, sequence features that can be uniquely identified byoligonucleotide probing, for example RFLP (restriction fragment lengthpolymorphism), or SSR markers (simple sequence repeat).

The efficiency with which endogenous homologous recombination occurs inthe cells of a given host varies from one class of cell or organism toanother. However the use of an efficient selection method or a sensitivescreening method can compensate for a low rate of recombination.Therefore, the basic tools for practicing the invention are available tothose of ordinary skill in the art for a wide range and diversity ofcells or organisms such that the successful application of such tools toany given host cell or organism is readily predictable. The compositionsand methods of the present invention can be designed to introduce atargeted mutation or genetic recombination into any host cell ororganism. The flexibility of the present invention allows for geneticmanipulation in order to create genetic models of disease or toinvestigate gene function.

The compositions and methods of the present invention can also be usedto effect targeted genetic recombination or mutation in a mammaliancell. In addition, a ZFN can be designed to cleave a particular gene orchromosomal locus, which is then injected into an isolated embryo priorto reimplantation into a female. ZFN-mediated DNA cleavage can occureither in the presence or absence of donor DNA. Offsprings can then bescreened for the desired genetic alteration.

The compositions and methods of the present invention can also be usedto accomplish germline gene therapy in mammals. In one embodiment, ZFNscould be designed to target particular genes of interest. Eggs and spermcould be collected and in-vitro fertilization performed. At the zygotestage, the embryo could be treated with both a ZFN designed to target aparticular sequence and a donor DNA segment carrying a sequence withoutthe deleterious mutation. The embryo could then be returned to a femaleor a uterine alternative for the rest of the gestational period. In aparticular embodiment, for example, the deleterious gene is the commoncystic fibrosis (CF) allele delta F508. ZFNs and donor DNA are usedaccording to the methods of the present invention in order to alleviatedisease caused by a mutant gene. According to the method, eggs and spermfrom known carrier parents are collected and in-vitro fertilized. Afterin-vitro fertilization, the zygote could be injected with ZFNs designedto target the delta F508 allele, and with donor DNA carrying thewild-type allele. The transformed zygote could then be reimplanted intothe mother. With the compositions and methods of the present invention,such gene replacement would allow the offspring and all descendants tobe free of the CF mutation.

In another embodiment, homologous recombination can be used as follows.First, a site for integration is selected within the host cell.Sequences homologous to those located upstream and downstream from theintegration site are then included in a genetic construct, flanking theselected gene to be integrated into the genome. Flanking, in thiscontext, simply means that target homologous sequences are located bothupstream (5′) and downstream (3′) of the selected gene. The construct isthen introduced into the cell, thus permitting recombination between thecellular sequences and the construct.

As a practical matter, the genetic construct will normally act as farmore than a vehicle to insert the gene into the genome. For example, itis important to be able to select for recombinants and, therefore, it iscommon to include within the construct a selectable marker gene. Themarker permits selection of cells that have integrated the constructinto their genomic DNA. In addition, homologous recombination may beused to “knock-out” (delete) or interrupt a particular gene. Thus,another approach for inhibiting gene expression involves the use ofhomologous recombination, or “knock-out technology”. This isaccomplished by including a mutated or vastly deleted form of theheterologous gene between the flanking regions within the construct.Thus, it is possible, in a single recombinational event, to (i) “knockout” an endogenous gene, (ii) provide a selectable marker foridentifying such an event and (iii) introduce a transgene forexpression.

The frequency of homologous recombination in any given cell isinfluenced by a number of factors. Different cells or organisms varywith respect to the amount of homologous recombination that occurs intheir cells and the relative proportion of homologous recombination thatoccurs is also species-variable. The length of the region of homologybetween donor and target affects the frequency of homologousrecombination events, the longer the region of homology, the greater thefrequency. The length of the region of homology needed to observehomologous recombination is also species specific. However, differencesin the frequency of homologous recombination events can be offset by thesensitivity of selection for the recombinations that do occur. It willbe appreciated that absolute limits for the length of the donor-targethomology or for the degree of donor-target homology cannot be fixed butdepend on the number of potential events that can be scored and thesensitivity of the selection for homologous recombination events. Whereit is possible to screen 10⁹ events, for example, in cultured cells, aselection that can identify 1 recombination in 10⁹ cells will yielduseful results. Where the organism is larger, or has a longer generationtime, such that only 100 individuals can be scored in a single test, therecombination frequency must be higher and selection sensitivity is lesscritical. The method of the present invention dramatically increases theefficiency of homologous recombination in the presence ofextrachromosomal donor DNA (see Examples). The invention can be mostreadily carried out in the case of cells or organisms that have rapidgeneration times or for which sensitive selection systems are available,or for organisms that are single-celled or for which pluripotent celllines exist that can be grown in culture and which can be regenerated orincorporated into adult organisms. Rapid generation time is theadvantage demonstrated for the fruit fly, Drosophila, in the presentinvention. The plant cells, Arabidopsis are one example of pluripotentcells that can be grown in culture then regenerated or incorporated intoan intact organism, tobacco are another. These cells or organisms arerepresentative of their respective classes and the descriptiondemonstrates how the invention can be applied throughout those classes.It will be understood by those skilled in the art that the invention isoperative independent of the method used to transform the organism.Further, the fact that the invention is applicable to such disparateorganisms as plants and insects demonstrates the widespreadapplicability of the invention to living organisms generally.

Nucleic Acid Delivery

Transformation can be carried out by a variety of known techniques whichdepend on the particular requirements of each cell or organism. Suchtechniques have been worked out for a number of organisms and cells, andcan be adapted without undue experimentation to all other cells. Stabletransformation involves DNA entry into cells and into the cell nucleus.For single-celled organisms and organisms that can be regenerated fromsingle-cells (which includes all plants and some mammals),transformation can be carried out in in vitro culture, followed byselection for transformants and regeneration of the transformants.Methods often used for transferring DNA or RNA into cells includeforming DNA or RNA complexes with cationic lipids, liposomes or othercarrier materials, micro-injection, particle gun bombardment,electroporation, and incorporating transforming DNA or RNA into virusvectors. Other techniques are well known in the art.

Examples of Some Delivery Systems Useful in Practicing the PresentInvention

Liposomal formulations: In certain broad embodiments of the invention,the oligo- or polynucleotides and/or expression vectors containing ZFNsand, where appropriate, donor DNA, may be entrapped in a liposome.Liposomes are vesicular structures characterized by a phospholipidbilayer membrane and an inner aqueous medium. Multilamellar liposomeshave multiple lipid layers separated by aqueous medium. They formspontaneously when phospholipids are suspended in an excess of aqueoussolution. The lipid components undergo self-rearrangement before theformation of closed structures and entrap water and dissolved solutesbetween the lipid bilayers. Also contemplated are cationic lipid-nucleicacid complexes, such as lipofectamine-nucleic acid complexes. Lipidssuitable for use according to the present invention can be obtained fromcommercial sources. Liposomes used according to the present inventioncan be made by different methods and such methods are known in the art.The size of the liposomes varies depending on the method of synthesis.

Microinjection: Direct microinjection of DNA into various cells,including egg or embryo cells, has also been employed effectively fortransforming many species. In the mouse, the existence of pluripotentembryonic stem (ES) cells that are culturable in vitro has beenexploited to generate transformed mice. The ES cells can be transformedin culture, then micro-injected into mouse blastocysts, where theyintegrate into the developing embryo and ultimately generate germlinechimeras. By interbreeding heterozygous siblings, homozygous animalscarrying the desired gene can be obtained.

Adenoviruses: Human adenoviruses are double-stranded DNA tumor viruseswith genome sizes of approximate 36 Kb. As a model system for eukaryoticgene expression, adenoviruses have been widely studied and wellcharacterized, which makes them an attractive system for development ofadenovirus as a gene transfer system. This group of viruses is easy togrow and manipulate, and they exhibit a broad host range in vitro and invivo. In lytically infected cells, adenoviruses are capable of shuttingoff host protein synthesis, directing cellular machineries to synthesizelarge quantities of viral proteins, and producing copious amounts ofvirus.

Particular advantages of an adenovirus system for delivering DNAencoding foreign proteins to a cell include (i) the ability tosubstitute relatively large pieces of viral DNA with foreign DNA; (ii)the structural stability of recombinant adenoviruses; (iii) the safetyof adenoviral administration to humans; and (iv) lack of any knownassociation of adenoviral infection with cancer or malignancies; (v) theability to obtain high titers of recombinant virus; and (vi) the highinfectivity of adenovirus.

In general, adenovirus gene transfer systems are based upon recombinant,engineered adenovirus which is rendered replication-incompetent bydeletion of a portion of its genome, such as E1, and yet still retainsits competency for infection. Sequences encoding relatively largeforeign proteins can be expressed when additional deletions are made inthe adenovirus genome. For example, adenoviruses deleted in both the E1and E3 regions are capable of carrying up to 10 kB of foreign DNA andcan be grown to high titers in 293 cells.

Other Viral Vectors as Expression Constructs: Other viral vectors may beemployed as expression constructs in the present invention. Vectorsderived from, for example, vaccinia virus, adeno-associated virus (MV),and herpes viruses may be employed. Defective hepatitis B viruses, maybe used for transformation of host cells. In vitro studies show that thevirus can retain the ability for helper-dependent packaging and reversetranscription despite the deletion of up to 80% of its genome.Potentially large portions of the viral genome can be replaced withforeign genetic material. The hepatotropism and persistence(integration) are particularly attractive properties for liver-directedgene transfer. The chloramphenicol acetyltransferase (CAT) gene has beensuccessfully introduced into duck hepatitis B virus genome in the placeof the viral polymerase, surface, and pre-surface coding sequences. Thedefective virus was cotransfected with wild-type virus into an avianhepatoma cell line, and culture media containing high titers of therecombinant virus were used to infect primary duckling hepatocytes.Stable CAT gene expression was suvsequently detected.

Non-viral Methods: Several non-viral methods are contemplated by thepresent invention for the transfer into a host cell of DNA constructsencoding ZFNs and, when appropriate, donor DNA. These include calciumphosphate precipitation, lipofectamine-DNA complexes, andreceptor-mediated transfection. Some of these techniques may besuccessfully adapted for in vivo or ex vivo use.

In one embodiment of the invention, the expression construct may simplyconsist of naked recombinant DNA. Transfer of the construct may beperformed by any of the DNA transfer methods mentioned above whichphysically or chemically permeabilize the cell membrane. For example,polyomavirus DNA in the form of CaPO₄ precipitates was successfullyinjected into liver and spleen of adult and newborn mice which thendemonstrated active viral replication and acute infection. In addition,direct intraperitoneal injection of CaPO₄ precipitated plasmidexpression vectors results in expression of the transfected genes.

Transformation of Plants: Transformed plants are obtained by a processof transforming whole plants, or by transforming single cells or tissuesamples in culture and regenerating whole plants from the transformedcells. When germ cells or seeds are transformed there is no need toregenerate whole plants, since the transformed plants can be growndirectly from seed. A transgenic plant can be produced by any meansknown in the art, including but not limited to Agrobacteriumtumefaciens-mediated DNA transfer, preferably with a disarmed T-DNAvector, electroporation, direct DNA transfer, and particle bombardment.Techniques are well-known to the art for the introduction of DNA intomonocots as well as dicots, as are the techniques for culturing suchplant tissues and regenerating those tissues. Regeneration of wholetransformed plants from transformed cells or tissue has beenaccomplished in most plant genera, both monocots and dicots, includingall agronomically important crops.

Screening for Mutations

Methods for genetic screening to accurately detect mutations in genomicDNA, cDNA or RNA samples may be employed, depending on the specificsituation. A number of different methods have been used to detect pointmutations, including denaturing gradient gel electrophoresis (“DGGE”),restriction enzyme polymorphism analysis, chemical and enzymaticcleavage methods, and others. The more common procedures currently inuse include direct sequencing of target regions amplified by PCR™ andsingle-strand conformation polymorphism analysis (“SSCP”). SSCP reliesupon the differing mobilities of single-stranded nucleic acid moleculesof different sequence on gel electrophoresis. Techniques for SSCPanalysis are well known in the art.

Another method of screening for point mutations is based on RNasecleavage of base pair mismatches in RNA/DNA and RNA/RNA heteroduplexes.As used herein, the term “mismatch” is defined as a region of one ormore unpaired or mispaired nucleotides in a double-stranded RNA/RNA,RNA/DNA or DNA/DNA molecule. This definition thus includes mismatchesdue to insertion/deletion mutations, as well as single and multiple basepoint mutations.

EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventors to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Induction of Targeted Mutations

Zinc Finger Design

A pair of ZFNs were designed and constructed for a chomomosomal targetlocus in the yellow (y) gene of Drosophila. Zinc fingers generally bindpreferentially to G-rich regions of DNA, and extensive study has beenperformed of fingers that bind all 5′-GNN-3′ triplets. Because thebinding sites must be in an inverted orientation with respect to eachother for effective cleavage by ZFNs, the chromosomal target locus ofDrosophila (y) was searched for inverted recognition sequences of theform (NNC)₃ . . . (GNN)₃. Such a site was identified in exon 2 with a6-bp separation between the component 9-mer recognition sites, which isthe optimal spacer for specific recognition and cleavage by ZFNs thathave no added linker or spacer between the binding and cleavage domains.The specific recognition sequences of the two ZFNs are described inBibikova et al. 2002, Genetics 161: 1169-1175. DNAs encoding zincfingers that recognize the DNA sequences, 5′-GCGGATGCG-3′ (SEQ ID NO:1)and 5′-GCGGTAGCG-3′ (SEQ ID NO:2), were obtained from Drs. David Segaland Carlos Barbas (Scripps Research Institute, La Jolla, Calif.). TheDNAs encoding the zinc fingers were then modified using mutagenic PCRprimers, and two sets of three zinc fingers each were produced: one,referred to as yA that recognizes one of the component 9-mers of the ygene target (5′-GTGGATGAG-3′ (SEQ ID NO: 3)), and another, referred toas yB, that recognizes the other component 9-mer of the y gene target(5′-GCGGTAGGC-3′ (SEQ ID NO: 4)). Two fingers were modified in yA, butonly one in yB. DNA encoding each of the resulting 3-finger sets of zincfingers were both cloned in frame with the FokI DNA cleavage domain inthe pET15b expression plasmid, with no intervening linker DNA betweenthe DNA recognition and cleavage domains. Both chimeric ZFN proteinswere expressed, purified by Ni-affinity chromatography, and tested forcleavage activity in vitro, using the pS/G plasmid, which carries thecomplete y gene. Together the two ZFNs made a single double strandedbreak (DSB) at the expected site in a 10.7-kb plasmid DNA carrying the ygene.

P Element Vectors and Transformation of Fly Larvae.

The yA and yB ZFN coding sequences were then cloned separately behindthe Drosophila Hsp70 heat shock promoter by insertion of ZFN DNA betweenthe BamHI and SalI sites of a modified phsp70 plasmid. A fragmentcarrying the heat shock promoter and ZFN DNA sequences was excised bypartial HindIII and complete ApaI digestion and cloned between thesesame endonuclease sites in the commercially available cloning vector,pBluescript. After verification of the sequence of the insert, it wasexcised by digestion with NotI and inserted into the ry+ P elementvector pDM30. The resulting yA and yB plasmids were injected separatelyinto v ry embryos, along with the P-transposase expression plasmidpπ25.lwc, and eclosing adults were mated to screen for ry+ germlinetransformants. The ry+ insertion was mapped to a specific chromosome formultiple independent transformants with each ZFN. Both balanced andhomozygous stocks were created for several lines carrying yA and yBwithout viability problems in most cases. Genes for the two ZFNs werebrought together (as described in the Examples below) with appropriatecrosses of mature flies, and the offspring were heat shocked 4 daysafter the initiation of mating by immersing the glass vials containingthe flies in a water bath at 35° for one hour. As adults eclosed theywere screened for evidence of somatic y mutations. Control vials fromcrosses involving each nuclease separately were subjected to the heatshock, and yA+yB flies that had not been heat shocked were alsoscreened. Recovery of germline mutants.

All flies emerging from the heat shock protocol and carrying both the yAand yB nucleases were mated to reveal potential germline mutations.Males were crossed with 2 or 3 attached-X [C(1)DX] females, and theresulting male offspring screened for yellow body color. Females werecrossed with 2 or 3 y (FM6) males, and the resulting offspring of bothgenders screened. Mutants were identified and all of them were malesthat had originated from male parents. These identified mutant maleoffspring were then crossed to C(1)DX females to produce additionalprogeny carrying the same mutation. DNA analysis.

The presence or absence of the target DNA was identified by DNAanalysis. Individual flies were homogenized in 100 μl of a 1:1 mixtureof phenol and grind buffer (7 M urea, 2% SDS, 10 mM Tris, pH 8.0, 1 mMEDTA, 0.35 M NaCl) preheated to 60°. Each sample was extracted with 50μl of chloroform, the organic phase back-extracted with 100 μl of grindbuffer, and the combined aqueous phases re-extracted with 50 μl ofchloroform. DNA was precipitated with ethanol and re-dissolved in 20 μlof 10 mM Tris, pH 8.5. A 600-bp DNA fragment was amplified by PCR withprimers flanking the yA+yB recognition site. The primers were called YF2(5′ATTCCTTGTGTCCAAAATMTGAC-3′ (SEQ ID NO:5)) and YR3(5′-AAAATAGGCATATGCATCATCGC3′ (SEQ ID NO:6)) For the larger deletions,YR3 was used in combination with a more distant sequence, YF1(5′ATTTTGTACATATGTTCTTMGCAG-3′ (SEQ ID NO:7)). Amplified fragments wererecovered after gel electrophoresis, and DNA sequences were determinedat the University of Utah DNA Sequencing Core Facility with an ABI3700capillary sequencer and the YR3 primer.

Induction of Targeted y Mutations Resulting From Double Stranded Breaksand Nonhomologous End Joining

The levels of expression of yA induced at 37° were found, in severalindependent transformants, to be lethal when applied at larval andembryonic stages. Moderating the heat shock to 35° allowed survival of agood proportion of the yA-carrying flies. The yB ZFN did not affectviability at any temperature tested.

After individual flies carrying the yA and yB nucleases on the samechromosome were crossed and their progeny heat-shocked, offspringdemonstrating y mosaic, as well as germline mutations were observed inmale offspring. In males (except following DNA replication), only simplereligation or NHEJ would be available to repair the damage after a DSB.In Drosophila, as in many other eukaryotes, NHEJ frequently producesdeletions and/or insertions at the joining site. Since the DSB istargeted to protein coding sequences in y+, most such alterations wouldlead to frameshifts or to deletion of essential codons, which can leadto a phenotype of patches of y mutant tissue.

Somatic yellow mosaics were identified in multiple yA+yB males. Most ofthe patches were in the distal abdominal cuticle and bristles, but someexamples in leg, wing and scutellar bristles were also observed. Noother phenotypic defects have been seen on a regular basis. Thefrequency of somatic mosaics was quite high. In pooled data from crossesinvolving a number of independent yA and yB lines, 105 of 228 candidatemales (46%) showed obvious y patches. For some yA+yB combinations thefrequency was greater than 80%. No yellow mosaics were observed incontrols with a single nuclease or without heat shock. This indicatesthat the yA+yB ZFNs are capable of inducing somatic mutations at theirdesignated target.

Characterization of Germline y Mutations.

To isolate germline y mutations, all yA+yB males from several heat shockexperiments were crossed to females carrying an attached-X chromosome[C(1)DX/Y], in order to produce male offspring that were known to onlyreceive their father's X chromosome. In total, 228 male fathers yielded5,870 sons; 26 of the male off-spring, from 13 different fathers, wereclearly y throughout their entire bodies. Thus, 5.7% of the yA+yB malefathers produced at least one germline mutant. Of the 13 fathers, 6 hadbeen identified as having y somatic patches, while the other 7 appearedto be entirely y+in diagnostic features. No y flies were isolated among7050 progeny of 125 heat-shocked yA+yB females crossed to y males. TheZFNs appear to be effective in inducing mutations via NHEJ mostefficiently in the male germline.

DNA was isolated from the 13 fathers identified above and 5 additionalmales in order to analyze each of them for the presence of the targetDNA. A 600-bp fragment including the expected cleavage site wasamplified by PCR. In three of the 18 male flies, the binding site forone of the primers had been deleted, and a new primer had to begenerated in order to accomplish amplification. This new primer waslocated at a more distant location. Sequence analysis of all fragmentsrevealed unique alterations precisely at the target site. Nine of thesequenced mutants had simple deletions; five had deletions accompaniedby insertions; and three were simple, short duplications. Three of thedeletions extended for hundreds of bps to one side of the target andthese were the three samples that required a new primer design. Theseare exactly the types of mutations that were expected to result fromNHEJ after cleavage by the yA+yB ZFNs, and they are very similar tothose produced after P element excision. Some of the frameshift ymutations created a stop codon within a short distance of thealteration, while one inserted an asparagine codon into the normalreading frame.

Targeted Cleavage and Mutagenesis.

This example demonstrated that ZFNs can be designed to produce DSBs intarget chromosomal locus in an exemplary genome in order to produce apermanent genetic alteration. The frequency of observed somatic mutationwas quite high, and the real number of somatic mosaics may be evenhigher, since y mutations have no effect on many visible features. Thiswas corroborated by the recovery of germline mutations fromphenotypically y+parents.

In this particular Example, germline mutations were recovered only inmales and at a lower frequency than somatic mosaics.

Example 2 ZFN-Induced Double Stranded Breaks Stimulate Targeted GeneticRecombination in the Presence of Homologous Donor DNA

Zinc Finger and Donor DNA Design

A pair of ZFNs were designed and constructed for a chromosomal targetlocus in the yellow (y) gene of Drosophila as described in Example 1.

In order to make an identifiable donor DNA for the Drosophila gene, y,the yA and yB recognition sites for the zinc fingers were replaced withtwo in-frame stop codons and an XhoI site. These changes were introducedby amplification with PCR primers carrying the desired sequence.Relative to the wild type y, 21 bp were deleted leaving only 3 bp of theyA recognition site, and a 9 bp replacement inserted the two in-framestop codons and inserted the XhoI site. This mutant (yM) carries a totalof 8 kb of homology to the y locus. It was inserted into a P elementvector and introduced into the fly genome. The yM sequence is flanked byrecognition sites for the FLP recombinase (FRT) and the meganucleaseI-SceI to permit excision and linearization of the donor. Generating alinear extrachromosomal donor DNA in situ by this means has been shownto enhance its effectiveness in recombination.

Experimental Design

The design of the targeted genetic recombination experiment is asfollows: The y⁺ target lies on the X chromosome. The transgenes for theyA and yB ZFNs are on one chromosome 2, while those for FLP and/orI-SceI (when present) are on the other chromosome 2. The donor DNA (yM)is located on chromosome 3 in a p-element vector that also carries thewhite gene (W+). Each of these inserted genes is under the control of aDrosophila HSP70 promoter. Upon heat-shock induction, the ZFNs will cuttheir target at y. This broken chromosome can be restored to wild type,or it can acquire a y mutation either by NHEJ or by homologousrecombination. When neither FLP nor I-SceI is present, the donor remainsintegrated. When FLP is expressed, the donor is excised as anextrachromosomal circle. When I-SceI is also expressed, it converts thedonor to an ends-out linear molecule which can recombine with thecleaved target locus. Experiments were also performed with linear donoronly in the absence of yA and yB (and therefor without cleavage of thetarget).

Larvae carrying single copies of these introduced DNA components wereheat-shocked at 35°, for one hour, 0-4 days after egg laying. Theexperiment contained five groups as exemplified below:

-   -   ND, no donor: yA+yB only;    -   ID, integrated donor: yA+yB+donor, no FLP or I-SceI;    -   CD, circular extrachromosomal donor: yA+yB+FLP+donor;    -   LD, linear extrachromosomal donor: yA+yB+FLP+I-SceI+donor;    -   DO, linear donor only: FLP+I-SceI+donor, but no ZFNs.

Adults emerging from the heat shock protocol were crossed to revealgermline y mutations. The frequencies of germline y mutations resultingfrom the heat-shock treatment are shown in Table 1 in column 3. Thefrequencies of mutation rose in both males and females in the presenceof the donor and the frequency increased further with extrachromosomaland linear DNA. With linear extrachromosomal DNA, nearly 20% of malesand 14% of females yielded at least one mutant offspring.

The y mutations were propagated in further crosses, chromosomal DNA wasrecovered. The frequency of germline y mutants and the proportion due toeither NHEJ or homologous recombination with the donor DNA wasdetermined by PCR amplification of 600 bp of DNA including the targetregion of the y gene followed by XhoI digestion of the amplifiedproduct. Products of homologous recombination between donor and targetwere recognized by XhoI digestion of the PCR fragment; some of these andmany of the XhoI-resistant products were sequenced. The latter showedsmall deletions and/or insertions and occasionally larger deletions, allof which are characteristic of NHEJ.

The fourth column of Table 1 reports the recovery of germline mutants asa percentage of all offspring. The fractions of those mutationsresulting from either NHEJ or homologous recombination with the donorrose as the donor DNA became more effective at participating inhomologous recombination: linear donor DNA being more effective thancircular donor DNA, which was more effective than integrated donor DNA.The integrated donor, located on chromosome 3, was not very effective inserving as a template for repair of the break at y and the majority ofrecovered mutations were due to NHEJ. The circular donor was much moreeffective and approximately 1/3 of all mutations were determined to bedue to gene replacements. With the linear donor, more than 2% of allsons of males were mutant, and 63% of these were products of homologousrecombination. In the female germline 73% of y mutations were homologousreplacements. Target cleavage by chimeric ZFNs stimulates targetedgenetic recombination substantially, and the most effective way tointegrate donor DNA into a host organism's genome is with linear donorDNA.

The ZFN-induced targeted genetic recombination results differ from thoseobtained without targeted cleavage in several respects. First, inducedmutations were found in both the male and female germlines, while onlyfemales had yielded good frequencies in previous trials by otherresearchers. Apparently the presence of a DSB in the target activatesrecombination processes in males that are not efficient on intactchromosomes. The lower targeting frequencies observed in females mayreflect the possibility of repairing the break by recombination with anuncut homologous X chromosome. Second, the overall frequency of inducedmutations was about 10-fold higher in males in the linear DNA andcircular DNA experiments than was seen earlier at y in females with anends-in donor: approximately 1/50 gametes, compared to 1/500 gametes.Even in the female germline, the frequency of ZFN-induced mutations was1/200 gametes, and ¾ of these were gene replacements. Thus, the presenceof a homologue donor does not preclude interaction with theextrachromosomal donor. Third, deletions and insertions due to NHEJ werealso observed, in addition to the targeted homologous recombinants. Suchproducts were not expected nor observed in the absence of targetcleavage.

Example 3 Expression of Chimeric ZFNs in Arabidopsis in Order toStimulate Induction of Targeted Mutations

Experimental Design

The method of the present invention will be used to target and knock outthe Arabidopsis TRANSPARENT TESTA GLABRA1 gene (TTG1, gene numberAT5G24520 (GenBank number AJ133743). An EST for this gene has beensequenced (GenBank numbers F20055, F20056). The gene encodes a proteincontaining WD40 repeats.

Two chimeric DNA constructs will be generated consisting of (1) nucleicacid sequence encoding the promoter region from the Arabidopsis HSP18.2gene and (2) nucleic acid sequence encoding zinc finger proteinsspecific for the TTG1 gene operatively linked to a nucleic acid sequenceencoding a non-specific endonuclease. The HSP18.2 promoter will conferexpression in Arabidopsis and gene expression will be controlled byheat-shocking the resulting plants. The chimeric genes will be referredto as HS::ZnTTG1 A and HS::ZnTTG1B. These two genes can be incorporatedinto the same Agrobacterium vector.

All of our experiments will be carried out using the model geneticorganism Arabidopsis thaliana, because of a number of desirable featuresof this system including small size, small genome, and fast growth. Attg1 mutant has a distinctive phenotype, making it an excellentexemplary model. For instance, ttg1 mutants are glabrous and mutantplants lack trichomes on leaves and stems. Trichomes are hair-likeoutgrowths from the epidermis.

Additionally, ttg1 mutant are defective in flavonoid production.Flavonoids are a complex class of compounds including purple anthocyaninpigments and tannins. TTG1 protein positively regulates synthesis of theenzyme dihydroflavonol reductase, which is required for production ofboth anthocyanins and tannins.

These ttg1 mutants also have a transparent testa or seed coat. In wildtype, the seed coat (inner layer of the inner integument) has dense,brown tannin and ttg1 mutants lack this pigment. As a consequence, theseed coat of seed collected from ttg1 mutants are transparent, and seedcollected from ttg1 mutants are yellow because the yellow embryos showthrough the transparent seed coat.

These ttg1 mutants also lack anthocyanins. In wild type, seedlings,stems, and leaves produce reddish/purple anthocyanin pigments,particularly under stress. These pigments are absent in ttg1 mutants.

Additionally, ttg1 mutants produce extra root hairs. In wild type, roothairs are produced only from trichoblast cells. In ttg1 mutants, bycontrast, root hairs are produced by both tricoblast cells andatrichoblast cells. The result is a root that appears more hairy.

The ttg1 mutants also fail to produce mucilage in the outer layer of theseed coat. Mucilage is a complex carbohydrate, sometimes called slimethat covers the seed coat. Lastly, the ttg1 mutants have altereddormancy and ttg1 seeds do not require drying out or cold treatments togerminate.

The presence of all seven characteristics makes visual screening forthis mutant genotype an easy task.

Design of Zinc Fingers

The TTG1 gene was scanned for sequences of the form: NNY NNY NNY NNNNNNRNN RNN RNN, where Y is either T or C, R is A or G, and N is any base.This identified sequences comprised of triplets that are initiated by anA or G in opposite orientation—i.e., on opposite strands—and separatedby exactly 6 bp. This has been shown to be a preferred structure forzinc finger nuclease recognition and cleavage.

The component triplets of the sequences identified in 1 were thenclassified according to whether there were zinc fingers that were knownto bind them specifically. Two sites in TTG1 were identified aspotential ZFN binding and cleavage sites: 5′-TCC GGT CAC AGA ATC GCC GTCGGA-3′ (SEQ ID NO:8), and 5′-ACT TCC TTC GAT TGG AAC GAT GTA3′ (SEQ IDNO:9) (at nucleotide 406 in the TTG1 sequence).

Zinc finger nucleases comprising a binding domain designed to bind thefirst of these sites will be constructed either by oligonucleotidesynthesis and extension, or by PCR with mutagenic primers. The resultingcoding sequences will be inserted into plasmids vectors in frame withthe FokI nuclease domain to create two ZFN coding sequences, ZnTTG1A andZnTTG1B. The encoded proteins will be expressed in E. coli and partiallypurified. The recovered ZFNs will be tested in vitro for the ability tocleave plasmid DNA encoding the TTG1 gene. Success in this assay will beevidenced by no cleavage by either ZFN alone, but cleavage at theexpected site by a mixture of the two ZFNs.

Transformation

The HS::ZnTTG1A and HS::ZnTTG1 B genes will be introduced into theArabidopsis genome using Agrobacterium-mediated transformation. To doso, the HS::ZnTTG1A and B genes will be inserted into an AgrobacteriumT-DNA transformation vector (pCAMBIA1380) that harbors a selectablehygromycin resistant marker. A pCAMBIA HS::ZnTTG1 clone will then beintroduced into Agrobacterium cells using standard Agrobacteriumtransformation procedures, and the HS::ZnTTG1A and HS::ZnTTG1B geneswill then be introduced into Arabidopsis plants using the standardfloral dip method.

Induction of Expression of ZFNs in a Host Cell

Seeds from the T1 generation will be collected from the dipped plants.In order to select for transformed seedlings, the T1 seeds will begerminated on agar plates containing the antibiotic hygromycin.Approximately four days after germination, the plates containing thegerminated seedlings will be wrapped in plastic wrap and immersed in 40°C. water for two hours to induce expression of the ZFN genes. Atapproximately two weeks following germination, the hygromycin resistanttransformed seedlings will be transferred to dirt.

Screening for Gene-Targeting Event

Screening Method 1: The HS::ZnTTG1 genes will be introduced intowild-type Arabidopsis plants and the T1 plants will be heated asdescribed above. At 1-2 weeks following heat treatment, a sample oftissue will be harvested from heat-treated plants and DNA extracted fromthis tissue. PCR amplification using 20 bp primers flanking the zincfinger target site (25 bp on each side of the target site) will beutilized to determine if the HS::ZnTTG1 gene is present. The PCR bandfrom control plants that were not heat treated should be approximately90 bp in size. PCR bands from the heat-treated plants should includesmaller products than 90 bp that result from the existence of deletionssurrounding the zinc finger target site. To verify the existence ofsmall deletions, we will clone and determine the DNA sequence of thesmaller PCR products.

Screening Method 2: The HS::ZnTTG1 A and HS::ZnTTG1 B genes will beintroduced into wild-type Arabidopsis plants and the T1 plants will beheat-treated as described above. The T1 plants will be grown tomaturity, allowed to self pollinate, and T2 seeds will be collected. TheT2 seeds will be grown on agar plates and they will be scored forseedling phenotypes including hairless leaves (glabrous phenotype),brighter leaves (anthrocyanin minus phenotype), and hairy roots, asdescribed above. Mutant plants will be transferred to dirt and grownfurther. Tissue from mutant plants will be harvested and DNA extractedin preparation for PCR-screening as described above. Briefly, PCR willbe performed with primers flanking the zinc finger target sites andsamples exhibiting approximately 90 bp products were not transformed,whereas those exhibiting products less than 90 bp were transformed. Thisis due to the existence of deletions surrounding the zinc finger targetsite. Additionally, small insertions or much larger deletions may bepresent around the zinc finger target site, as well. To verify theexistence of these occurrences, we will clone and determine the DNAsequence of the smaller PCR products.

Screening Method 3: The HS::ZnTTG1 A and HS::ZnTTG1 B genes will beintroduced into heterozygous ttg1 mutants (i.e., genotype ttg1/TTG1).The male sterile1 (ms1) plants will be introduced to the Agrobacteriumsolution (note: the ms1 and ttg1 loci are linked, 6 cM apart onchromosome 5). The dipped plants then will be pollinated with pollenfrom homozygous ttg1-1 plants. The crossed plants will be allowed tomature, the resultant T1/F1 seeds collected, and the T1/F1 seeds allowedto germinate in the presence of hygromycin. Surviving T1/F1 seedlingswill contain the HS::ZnTTG1 transgene and will be heterozygous at thettg1 locus (i.e., genotype MS1-ttg1-1/ms1-TTG1). The T1/F1 plants willbe heat-shocked as described above. In a subset of cells, the wild-typeallele will be knocked out, resulting in a sector of homozygous ttg1(i.e., genotype ttg1-1/ttg1-ko) cells. These mutant sectors will bedetectable (and, thus, a targeted genetic recombination event) byvisualizing several phenotypes, such as hairless leaves (glabrousphenotype), brighter leaves (anthocyanin minus phenotype), and yellowseeds (transparent testa phenotype). Tissue will be collected frommutant sectors and targeting verified using the PCR-cloning-sequencingstrategy discussed above. From the mutant sectors, T2 seeds will becollected and grown into T2 plants. In the T2 generation, the phenotypewill be verified: plants homozygous for the knockout allele (i.e.,ttg1-ko) also will be homozygous for the ms1 mutation and, thus, will bemale sterile (i.e., genotype ms1-ttg1-ko/ms1-ttg1-ko). Tissue from thedouble mutants (phenotypically ttg1 and ms1) will be harvested andverified for targeting using the PCR-cloning-sequencing strategydiscussed above.

Example 4 Promotion of Recombination by ZFNs in Tobacco Cells

Experimental Design

The method of the present invention was used to repair artificiallyintroduced target genes in tobacco cells via recombination. A targetgene was constructed that conferred both selectable and screenablephenotypes to plant cells. The target gene was then renderednon-functional by mutation and stably transformed into tobacco plants. Azinc finger array that recognized the target gene was then fused to thenuclease domain of FokI. A donor DNA molecule was also constructed thatcould correct the mutation in the target gene if recombination occurred.

Creation of a Zinc Finger Nuclease

A zinc finger domain from the mouse transcription factor, Zif268, wasused. DNA encoding the Zif268 DNA binding domain was fused to sequencesencoding FokI nuclease to create a Zif268::FokI fusion. The Zif268 DNAbinding domain and FokI restriction endonuclease domain were synthesizedusing PCR and overlapping oligonucleotides. The sequences of both weremodified to match codon bias for dicot plants. The two domains werejoined to yield a Zif268::FokI fusion. The Zif268::FokI fusion wasmodified for expression in plants by placing it behind a nopalinesynthase (NOS) promoter and upstream of the NOS transcriptionalterminator. A nuclear localization signal and an AcV5 epitope tag wereadded to the N-terminus of the Zif268::FokI fusion to direct the proteinto the plant nucleus and to enable detection by immunoblot analysis.

Creation of a Target Gene

To test whether the Zif268::FokI fusion protein could cleave plantchromosomes and enhance recombination, a target gene with Zif268 bindingsites was created. The target gene was a translational fusion betweenthe β-glucuronidase (GUS) and neomycin phosphotransferase (NPTII) genes.A functional GUS::NPTII fusion protein confers kanamycin resistance, andcells expressing GUS activity turn blue when incubated in appropriatesubstrates. The GUS gene was placed downstream of a constitutivepromoter. An artificial intron (AI) was inserted into the GUS codingsequence to aid in reverse transcription dependent polymerase chainreaction (RT-PCR) experiments to measure GUS expression; RT-PCR productsproduced from spliced mRNAs would lack the intron allowing them to bedistinguished from contaminating genomic DNA. The GUS coding sequencewas fused to NPTII and placed upstream of the NOS terminator. The targetgene was then rendered non-functional by deleting 600 bases of sequencesencompassing the coding region for the active sites of both GUS andNPTII. A recognition site for Zif268 was inserted at the site ofdeletion. The mutated GUS::NPTII gene was then stably transformed intotobacco cells to generate transgenic tobacco callus. Twelve independenttransgenic plants were regenerated and the primary transformants, whichcarried the mutant GUS::NPTII gene by PCR analysis, were used for allsubsequent experiments. The function of the target gene could berestored by recombination, thereby enabling one to test whetherZif268::FokI could cleave the target gene in vivo and enhancefrequencies of recombination.

Experimental Results

Twelve independent transgenic plants were regenerated from tobaccocallus. These primary transformants were used in all subsequentexperiments. PCR analysis confirmed that all twelve plants carried themutant GUS::NPTII gene. To test whether the GUS::NPTII gene was in achromosomal environment conducive to transcription, RT-PCR experimentswere conducted with RNA isolated from leaf tissue. The primers were oneither side of the AI, and therefore the PCR product could distinguishbetween RNA and contaminating genomic DNA (or unspliced mRNA). Nine ofthe twelve transgenic plants showed strong expression of the GUS::NPTIImarker. Three of the plants gave ambiguous results, and displayed bandscorresponding to sizes predicted for both RNA and genomic DNA.

The Zif268::FokI fusion was then cloned into a vector for expression inE. coli. After induction of expression, presence of the protein wasverified by immunoblot analysis. The activity of the fusion protein wastested in vitro by incubating a few microliters of E coli lysate with atarget vector containing a Zif268 recognition site. The target vectorwas cleaved efficiently.

Construction of the Donor DNA Molecule

To correct the defective GUS::NPTII gene, a donor DNA molecule wascreated with wild type sequences for the active sites of GUS and NPTII.Upon recombination with the defective chromosomal target gene, the donormolecule would restore function. The donor molecule shared 4.3 kb ofhomology with the chromosomal target and included an additional 600bases that encoded the active sites of GUS and NPTII. To minimize thechance that integration into the plant chromosome by illegitimaterecombination would confer kanamycin resistance or GUS activity, thedonor molecule lacked a promoter and part of the coding sequence forGUS. In addition, to help in the molecular characterization of putativerecombinants, the donor DNA molecule had a diagnostic restrictionendonuclease site that was absent from the GUS::NPTII construct.

Selecting Antibiotic Resistant Cells after Introduction of Zinc FingerNucleases and Donor DNA.

Tobacco protoplasts were generated from transgenic plants harboring themutant GUS::NPTII gene. Ten plants were used in the study (designated 1,3-6 and 8-12); two independent experiments were carried out with plants#9 and #12 (Table 2). For each electroporation experiment, 1×10⁶protoplasts were used. Plasmid DNAs were linearized with a restrictionenzyme prior to electroporation. The protoplasts were electroporatedwith either the functional GUS::NPTII fusion, donor DNA, DNA encodingthe Zif268:FokI fusion protein, or both. The ZFN plasmid was provided ina 5:1 molar excess relative to the donor DNA, using a maximum of 35 μgof DNA. The amounts of ZFN and donor DNA were kept constant betweencontrol and test experiments.

After electroporation, the cells were allowed to recover for six days oncallus-inducing media. Kanamycin was then added to the media at thelevel of 50 μg/ml. After approximately 20 days, the number of kanamycinresistant cells was counted (Table 2). Each resistant callus was stainedfor GUS activity. In no case was GUS activity observed in controlexperiments using only the donor DNA or DNA encoding the ZFN.

Molecular Characterization of Potential Recombination Events

In the putative recombinants, the target gene was characterized by PCRusing a primer pair designed to amplify the region spanning the deletionin the GUS::NPTII gene. To prevent amplification of donor DNA moleculesthat integrated into chromosomes by illegitimate recombination, oneprimer was specific to the target gene and the other was complementaryto sequences in both the target and donor DNA. Additionally, acommercially available, high fidelity PCR kit (Roche Diagnostics,Indianapolis, Ind.) was used that contains a detergent to prevent strandtransfers between the target gene and donor DNA during amplification.

DNA was prepared from Kan^(r) calli derived from experiments performedwith plant #6. These included seven Kan^(r) GUS⁺ calli and five thatwere Kan^(r) GUS⁻. These twelve calli were generated by electroporationof both the donor and ZFN-encoding DNA. Two PCR reactions were conductedwith each DNA preparation, and the results of both reactions wereconsistent. Six of the Kan^(r) GUS⁺ calli produced PCR productsconsistent with having sustained a homologous recombination event. Thefailure of the remaining GUS positive callus to produce a PCR productcould be due to failure of the PCR reaction or to a rearrangement of thetarget gene. Three of the five Kan^(r) GUS⁻ calli produced PCR productsof the size predicted for an intact chromosomal target gene (i.e. onethat has not undergone recombination). The two Kan^(r) GUS⁻ calli thatdid not produce a PCR product may have sustained deletions thatencompass one or both primer binding sites.

PCR products from the six Kan^(r) GUS⁺ positive calli were sequenced toverify that they originated from a target gene whose function had beenreconstituted by recombination. The PCR products had diagnostic sequencemarkers indicating that they were derived from the chromosomal targetgene. They also had the internal restriction endonuclease siteengineered into the donor DNA molecule.

Overall Results

Approximately 1 in 10 plant cells that took up DNA were found to havesustained a targeted recombination event. This frequency ofrecombination is greater than any previously reported in plants.

Control Experiments

Illegitimate recombination was monitored by electroporating tobaccoprotoplasts with the functional GUS::NPTII reporter (Table 2,“Functional”). Frequency of NPTII resistance approximated 10⁻³ oftreated cells (4.5×10⁻³).

The phenotypic consequence of integration of donor DNA was monitored incontrol experiments where tobacco protoplasts were electroporated withonly the donor DNA. Kanamycin resistance was observed at a frequencyapproximating ₁₀ ⁻⁴ of treated cells (3.2×10⁻⁴). Comparing this value tothe frequency of illegitimate recombination, the lack of a promoter onthe donor DNA caused a 10-fold reduction in kanamycin resistance. Noneof the Kan^(r) cells were positive for GUS activity. The lack ofKan^(r)GUS⁺ cells in experiments using only the donor DNA suggests thatfrequencies of unassisted recombination at the chromosomal GUS::NPTIIgene are less than the total number of treated protoplasts (<1.2×10⁻⁷).

A second control experiment was conducted wherein tobacco protoplastswere transformed with DNA encoding only the Zif268::FokI fusion protein.No Kan^(r)GUS⁺ calli were recovered, indicating that the Zif268::FokIfusion by itself did not confer a phenotype consistent with homologousrecombination. The Zif268::FokI construct also did not affect cellviability, because protoplast plating efficiencies did not differ intreatments with or without the ZFN construct.

Zif268::FokI Fusion and Donor DNA

In contrast to control experiments, electroporation of tobaccoprotoplasts with both the Zif268::FokI fusion and donor DNA gave rise tolarge numbers of kanamycin resistant cells—at frequencies more than3-fold higher than when donor DNA was used alone. A large number of thekanamycin resistant cells were also GUS⁺. Since no GUS⁺ cells wereobtained when the donor DNA and ZFN-encoding DNA were used separately,the number of Kan^(r)GUS⁺ cells can be taken as the frequency ofhomologous recombination. This frequency is 9.1×10⁻⁵.

Frequencies of recombination are typically reported as a ratio ofnon-homologous to homologous events. In cells treated with thefunctional GUS::NPTII reporter, the frequency of Kan^(r)GUS⁺ was1.0×10⁻³. Since this is a measure of the frequency of non-homologous orillegitimate recombination, the ratio of non-homologous to homologousevents, therefore, can be expressed as 1.0×10⁻³ divided by 9.1×10⁻⁵.This number approximates 10^(−1.) In other words, approximately 1 in 10plant cells that took up DNA underwent a homologous recombination event.The kanamycin resistance marker was deemed to be more sensitive formeasuring frequencies of recombination. However, in calculating therecombination frequency with the kanamycin marker, the number ofbackground events caused by the donor DNA alone must be taken intoaccount. This calculation is presented in Table 2. The homologousrecombination frequency based on the kanamycin maker approximates7.8×10⁻⁴. When compared to the frequency of kanamycin resistanceobtained through illegitimate recombination using the functional geneconstruct (4.5×10⁻³), it can be concluded that approximately 1 in 6cells that took up DNA underwent homologous recombination whichconferred kanamycin resistance.

Enhancements of recombination in plants due to chromosome breakage havepreviously been reported, for example through the use of restrictionendonucleases (1-SceI). The enhancement of recombination by ZFNs is aleast one order of magnitude higher than these previous reports.

All of the COMPOSITIONS, METHODS and APPARATUS disclosed and claimedherein can be made and executed without undue experimentation in lightof the present disclosure. While the compositions and methods of thisinvention have been described in terms of preferred embodiments, it willbe apparent to those of skill in the art that variations may be appliedto the COMPOSITIONS, METHODS and APPARATUS and in the steps or in thesequence of steps of the methods described herein without departing fromthe concept, spirit and scope of the invention. More specifically, itwill be apparent that certain agents that are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims. TABLE 1 Recovery ofgermline y mutations 1 2 3 4 Donor # Screened # Giving y Total yFemales: ND 125 0 (0%) 0 ID 188 9 (4.8%) 15 (0.16%) CD 309 31 (10%) 59(0.38%) LD 503 68 (13.5%) 135 (0.54%) DO 158 1 (0.6%) 2 (0.02%) Males:ND 228 13 (5.7%) 24 (0.42%) ID 218 24 (11%) 40 (0.73%) CD 261 49 (19%)104 (1.59%) LD 522 94 (18%) 292 (2.24%) DO 177 1 (0.6%) 1 (0.02%)

TABLE 2 ZFN-Mediated Recombination in Tobacco Protoplasts Donor ZFNDonor & Functional DNA Only DNA Only ZFN DNA Plant ID Kan^(r) GUS⁺Kan^(r) GUS⁺ Kan^(r) GUS⁺ Kan^(r) GUS⁺  1 6080 640 292 0 0 NA 1150 85  310320 1720 459 0 0 NA 2040 0  4 1520 640 417 0 0 NA 975 75  5 1400 360212 0 0 NA 442 0  6 8600 1320 356 0 0 NA 1387 99  8 2320 840 82 0 0 NA1088 88  9a 3360 1000 618 0 0 NA 1330 186  9b 3520 920 181 0 0 NA 743260 10 2360 840 317 0 0 NA 751 0 11 5480 1400 326 0 0 NA 1528 109 12a4400 1400 399 0 1 0 1044 87 12b ND — 220 0 0 NA 716 104 Average 4487.271007.27 323.25 0.08 1099.5 91.08 Frequency × 10⁻⁵ 448.72 100 32.32 0.008109.95 9.1Each experiment used 1 × 10⁶ protoplastsAn average estimate of the homologous recombination frequency based onthe kanamycin resistance marker is: 1099.5 − 323.25/10⁵ = 77.62 × 10⁻⁵

1. A method of targeted genetic recombination in a host cell comprising:introducing into the host cell a nucleic acid molecule encoding a ZincFinger Nuclease (ZFN) targeted to a chosen host target locus; inducingexpression of the ZFN within the host cell; and identifying arecombinant host cell in which the selected host DNA sequence exhibits amutation at the host target locus.
 2. The method of claim 1 wherein themutation is selected from the group consisting of a deletion of geneticmaterial, an insertion of genetic material, and both a deletion and aninsertion of genetic material.
 3. (canceled)
 4. (canceled)
 5. The methodof claim 1 further comprising introducing donor DNA into the host cell.6. The method of claim 5 wherein the donor DNA provides a gene sequencethat encodes a product to be produced in the host cell.
 7. The method ofclaim 6 wherein the donor DNA provides a gene sequence that encodes aproduct selected from the group consisting of pharmaceuticals, hormones,proteins, nutriceuticals and chemicals.
 8. The method of claim 1 whereinthe host cell is selected from the group consisting of a single celledorganism, a cell from a multicellular organism, and an oocyte. 9.(canceled)
 10. (canceled)
 11. The method of claim 8 wherein the hostcell is an insect cell.
 12. The method of claim 11 wherein the insect isa member of an order selected from the group Coleoptera, Diptera,Hemiptera, Homoptera, Hymenoptera, Lepidoptera, or Orthoptera.
 13. Themethod of claim 12 wherein the insect is selected from the groupconsisting of a fruit fly, a mosquito, and a medfly.
 14. (canceled) 15.The method of claim 8 wherein the multicellular organism is a plant. 16.The method of claim 15 wherein the plant is selected from the groupconsisting of a monocotyledon and a dicotyledon.
 17. The method of claim16 wherein the plant is selected from the group consisting of maize,rice, wheat, potato, soybean, tomato, tobacco, members of the Brassicafamily, and Arabidopsis.
 18. (canceled)
 19. (canceled)
 20. The method ofclaim 15 wherein the plant is a tree.
 21. The method of claim 8 whereinthe multicellular organism is a mammal.
 22. The method of claim 21wherein the mammal is selected from the group consisting of mouse, rat,pig, sheep, cow, dog and cat.
 23. The method of claim 8 wherein themulticellular organism is a bird.
 24. The method of claim 23 wherein thebird is selected from the group consisting of chicken, turkey, duck andgoose.
 25. The method of claim 8 wherein the multicellular organism is afish.
 26. The method of claim 25 wherein the fish is selected from thegroup consisting of a zebrafish, trout and salmon.
 27. The method ofclaim 8 wherein the mutation occurs in a cell selected from the groupconsisting of a germ line cell of a single celled organism, a germ linecell of a multicellular organism, a somatic cell of a single celledorganism and a somatic cell of a multicellular organism.
 28. (canceled)29. A method of targeted genetic recombination in a host cellcomprising: selecting a zinc finger DNA binding domain thatpreferentially binds to a specific host target locus; selecting a DNAcleavage domain that cleaves double-stranded DNA when operatively linkedto said binding domain and introduced into the host cell; selecting acontrol element that induces expression in the host cell; operativelylinking the DNA encoding the binding domain and the cleavage domain andsaid control element to produce a DNA construct; introducing said DNAconstruct into a target host cell; and identifying at least one hostcell exhibiting recombination at the target locus in the host DNA. 30.The method of claim 29 further comprising introducing donor DNA into thehost cell.
 31. The method of claim 30 wherein the donor DNA provides agene sequence that encodes a product to be produced in the host cell.32. The method of claim 31 wherein the donor DNA provides a genesequence that encodes a product selected from the group consisting ofpharmaceuticals, hormones, proteins, nutriceuticals and chemicals. 33.The method of claim 29 wherein the DNA binding domain is comprised ofthree zinc fingers.
 34. The method of claim 29 wherein the zinc fingersare selected from the group consisting of Cys₂His₂ zinc fingers.
 35. Themethod of claim 29 wherein the cleavage domain is selected from thegroup consisting of Type II restriction endonucleases.
 36. The method ofclaim 29 wherein the Type II restriction endonuclease is FokI.
 37. Themethod of claim 29 wherein the control element is selected from thegroup consisting of heat-shock inducible control elements. 38.(canceled)
 39. The method of claim 29 wherein the host cell is a singlecelled organism, a cell from a multicellular organism, a gamete cell, oran oocyte.
 40. (canceled)
 41. The method of claim 30 wherein the targetcell is a gamete cell of a host organism.
 42. The method of claim 29wherein the host cell is an insect cell.
 43. (canceled)
 44. (canceled)45. The method of claim 42 wherein the insect is a member of an orderselected from the group consisting of Coleoptera, Diptera, Hemiptera,Homoptera, Hymenoptera, Lepidoptera, and Orthoptera.
 46. The method ofclaim 45 wherein the insect is selected from the group consisting of afruit fly, a mosquito, and a medfly.
 47. (canceled)
 48. The method ofclaim 39 wherein the multicellular organism is a plant.
 49. The methodof claim 48 wherein the plant is selected from the group consisting of amonocotyledon and a dicotyledon.
 50. The method of claim 49 wherein theplant is selected from the group consisting of maize, rice, wheat,potato, soybean, tomato, tobacco, members of the Brassica family, andArabidopsis.
 51. (canceled)
 52. (canceled)
 53. The method of claim 48wherein the plant is a tree.
 54. The method of claim 39 wherein themulticellular organism is a mammal.
 55. The method of claim 54 whereinthe mammal is selected from the group consisting of mouse, rat, pig,sheep, cow, dog and cat.
 56. The method of claim 39 wherein themulticellular organism is a bird.
 57. The method of claim 56 wherein thebird is selected from the group consisting of chicken, turkey, duck andgoose.
 58. The method of claim 39 wherein the multicellular organism isa fish.
 59. The method of claim 58 wherein the fish is selected from thegroup consisting of a zebrafish, trout and salmon.
 60. The method ofclaim 39 wherein the multicellular organism is a fungus.
 61. The methodof claim 39 wherein the recombination occurs in a cell selected from thegroup consisting of a germ line cell of a single celled organism, a germline cell of a multicellular organism, a somatic cell of a single celledorganism and a somatic cell of a multicellular organism.
 62. The methodof claim 30 wherein the DNA binding domain is comprised of three zincfingers.
 63. The method of claim 30 wherein the cleavage domain isselected from the group consisting of Type II restriction endonucleases.64. The method of claim 30 wherein the control element is selected fromthe group consisting of heat-shock inducible control elements. 65.(canceled)
 66. The method of claim 30 wherein the host cell is selectedfrom the group consisting of a single celled organism, a cell from amulticellular organism, and an oocyte.
 67. (canceled)
 68. The method ofclaim 66 wherein the host cell is an insect cell.
 69. The method ofclaim 68 wherein the insect is a member of an order selected from thegroup consisting of Coleoptera, Diptera, Hemiptera, Homoptera,Hymenoptera, Lepidoptera, and Orthoptera.
 70. The method of claim 69wherein the insect is selected from the group consisting of a fruit fly,a mosquito, and a medfly.
 71. (canceled)
 72. The method of claim 66wherein the multicellular organism is a plant.
 73. The method of claim72 wherein the plant is selected from the group consisting of amonocotyledon and a dicotyledon.
 74. (canceled)
 75. (canceled)
 76. Themethod of claim 75 wherein the plant is selected from the groupconsisting of maize, rice, wheat, potato, soybean, tomato, tobacco,members of the Brassica family, and Arabidopsis.
 77. The method of claim72 wherein the plant is a tree.
 78. The method of claim 66 wherein themulticellular organism is a mammal.
 79. The method of claim 78 whereinthe mammal is selected from the group consisting of mouse, rat, pig,sheep, cow, dog and cat.
 80. The method of claim 66 wherein themulticellular organism is a bird.
 81. The method of claim 80 wherein thebird is selected from the group consisting of chicken, turkey, duck andgoose.
 82. The method of claim 66 wherein the multicellular organism isa fish.
 83. The method of claim 82 wherein the fish is selected from thegroup consisting of a zebrafish, trout and salmon.
 84. The method ofclaim 66 wherein the multicellular organism is a fungus.
 85. The methodof claim 66 wherein the recombination occurs in a cell selected from thegroup consisting of a germ line cell of a single celled organism, a germline cell of a multicellular organism, a somatic cell of a single celledorganism and a somatic cell of a multicellular organism.
 86. The methodof claim 30 wherein the DNA construct further comprises DNA encoding oneor more selectable markers.
 87. The method of claim 86 wherein theselectable marker provides positive selection for cells expressing themarker selected from the group consisting of positive selection,negative selection and both positive and negative selection. 88.(canceled)
 89. (canceled)
 90. The method of claim 8 wherein themulticellular organism is a fungus.
 91. A method of targeted geneticrecombination in a plant cell comprising: introducing into the plantcell a nucleic acid molecule encoding a Zinc Finger Nuclease (ZFN)targeted to a chosen host target locus; inducing expression of the ZFNwithin the plant cell; and identifying a recombinant plant cell in whichthe selected plant DNA sequence exhibits a mutation at the plant targetlocus.
 92. The method of claim 91 wherein the mutation is selected fromthe group consisting of a deletion of genetic material, an insertion ofgenetic material, and both a deletion and an insertion of geneticmaterial.
 93. The method of claim 91 further comprising introducingdonor DNA into the plant cell.
 94. The method of claim 93 wherein thedonor DNA provides a gene sequence that encodes a product to be producedin the plant cell.
 95. The method of claim 94 wherein the donor DNAprovides a gene sequence that encodes a product selected from the groupconsisting of pharmaceuticals, hormones, proteins, nutriceuticals orchemicals.
 96. The method of claim 91 wherein the plant is selected fromthe group consisting of a monocotyledon and a dicotyledon.
 97. Themethod of claim 96 wherein the plant is selected from the groupconsisting of maize, rice, wheat, potato, soybean, tomato, tobacco,members of the Brassica family, and Arabidopsis.
 98. The method of claim91 wherein the plant is a tree.
 99. A method of targeted geneticrecombination in a plant cell comprising: selecting a zinc finger DNAbinding domain that preferentially binds to a specific plant targetlocus; selecting a DNA cleavage domain that cleaves double-stranded DNAwhen operatively linked to said binding domain and introduced into theplant cell; selecting a control element that induces expression in theplant cell; operatively linking the DNA encoding the binding domain andthe cleavage domain and the control element to produce a DNA construct;introducing said DNA construct into a target plant cell; and identifyingat least one plant cell exhibiting recombination at the target locus inthe plant DNA.
 100. The method of claim 99 further comprisingintroducing donor DNA into the plant cell.
 101. The method of claim 100wherein the donor DNA provides a gene sequence that encodes a product tobe produced in the plant cell.
 102. The method of claim 101 wherein thedonor DNA provides a gene sequence that encodes a product selected fromthe group consisting of pharmaceuticals, hormones, proteins,nutriceuticals and chemicals.
 103. The method of claim 99 wherein theDNA binding domain is comprised of three zinc fingers.
 104. The methodof claim 103 wherein the zinc fingers are selected from the groupconsisting of Cys₂His₂ zinc fingers.
 105. The method of claim 99 whereinthe cleavage domain is selected from the group consisting of Type IIrestriction endonucleases.
 106. The method of claim 105 wherein the TypeII restriction endonuclease is FokI.
 107. The method of claim 99 whereinthe control element is selected from the group consisting of heat-shockinducible control elements.
 108. The method of claim 99 wherein theplant is selected from the group consisting of a monocotyledon and adicotyledon.
 109. The method of claim 108 wherein the plant is selectedfrom the group consisting of maize, rice, wheat, potato, soybean,tomato, tobacco, members of the Brassica family, and Arabidopsis. 110.The method of claim 99 wherein the plant is a tree.
 111. The method ofclaim 99 wherein the DNA binding domain is comprised of three zincfingers.
 112. The method of claim 99 wherein the DNA construct furthercomprises DNA encoding one or more selectable markers.
 113. The methodof claim 112 wherein the selectable marker provides positive selectionfor cells expressing the marker selected from the group consisting ofpositive selection, negative selection and both positive and negativeselection.